Synthesis, molecular docking analysis, molecular dynamic simulation, ADMET, DFT, and drug likeness studies: Novel Indeno[1,2-b]pyrrol-4(1H)-one as SARS-CoV-2 main protease inhibitors

Background The COVID-19 pandemic began in 2019 as a result of the advent of a novel coronavirus, SARS-CoV-2. At present, there are a limited number of approved antiviral agents for the treatment of COVID-19. Remdesivir, Molnupiravir, and Paxlovid have been approved by the FDA to treat COVID-19 infections. Research has shown that the main protease enzyme (Mpro) of SARS-CoV-2 plays a crucial role in the enzymatic processing of viral polyproteins. This makes Mpro an interesting therapeutic target for combating infections caused by emerging coronaviruses. Methods The pharmacological effects of pyrroles and their derivatives have a wide range of applications. In our study, we focused on synthesizing nine novel derivatives of 2-arylamino-dihydro-indeno[1,2-b] pyrrol-4(1H)-one, with a particular emphasis on their antiviral properties. Using in silico studies involving molecular docking and DFT analyses in the gas phase using the B3LYP/6-31++G(d,p) basis set, we studied these compounds with respect to their interactions with the Mpro of SARS-CoV-2. The results of the docking analysis revealed that the synthesized compounds exhibited favorable inhibitory effects. Notably, compound 5f demonstrated the highest effectiveness against the target protein. Furthermore, the pharmacokinetic and drug-like properties of the synthesized derivatives of 2-arylamino-dihydroindeno[1,2-b] pyrrol-4(1H)-one indicated their potential as promising candidates for further development as inhibitors targeting SARS-CoV-2. However, it is imperative to determine the in vitro efficacy of these compounds through comprehensive biochemical and structural analyses.


Introduction
In 2019, an outbreak of respiratory illness with distinct pneumonia symptoms surfaced and swiftly disseminated worldwide, leading the World Health Organization to designate it a pandemic [1].The disease, which has been named COVID-19, is caused by a previously unknown coronavirus that has been identified as SARS-CoV-2.Contracting SARS-CoV-2 leads to respiratory symptoms resembling mild to moderate influenza, along with severe lung damage and the potential for multiple organ failure, ultimately resulting in death.The ubiquitous spread of SARS-CoV-2 and the constant occurrence of COVID-19 outbreaks pose a significant threat to global public health.There are currently a limited number of antiviral drugs approved for treating COVID-19 patients.Remdesivir, Molnupiravir, and Paxlovid are FDA-approved drugs that have been used for the treatment of COVID-19 [2], Fig 1.
It is anticipated that the drugs will diminish hospitalizations and fatalities linked to COVID-19.Nevertheless, as the pandemic progressed, new SARS-CoV-2 strains like Delta and Omicron emerged.Analyses indicated the Delta variant confers heightened disease severity relative to the ancestral strain, while Omicron displays moderately elevated transmissibility.The recently discovered mutations exhibit genomic anomalies that might possibly result in atypical epidemiological characteristics and the capacity to elude the immune system in the SARS-CoV-2 virus.This might potentially impede the efficacy of the methods of diagnosis and therapy and perhaps instigate further outbreaks [3].The SARS-CoV-2 virus's different strains are principally characterized by alterations in the spike protein of the virus.Nonetheless, there is still the potential for mutations in other components of the virus.A recent research has shown that the spike protein and chymotrypsin-like protease (3CL pro ), also known as the M pro , have a vital function in the replication and transcription of the virus.Therefore, they are a desirable target for the creation of therapeutic therapies against SARS-CoV-2 [4].Consequently, drugs that impede the functioning of this protease have the potential to stop viral replication.N-heterocyclic scaffolds are crucial in the study and advancement of drugs since they form the primary structural element of many physiologically active compounds.Their capability to interact with various cellular systems in living organisms has greatly contributed to their versatility.These compounds possess the capacity to disrupt a host factor involved in the replication process, impede the generation of new viral proteases, suppress an enzyme generated by the virus, or obstruct the entrance of the virus into host cells in patients [5][6][7][8].Considering these factors and as part of our ongoing research to develop novel and bioactive N- heterocycles, we focused on synthesizing a new series of dihydroindeno [1,2-b]pyrrol-4(1H)one derivatives.The aim of our study was to demonstrate the antiviral activity of these compounds against the M pro of SARS-CoV-2 by using in silico methods.In fact, studies show that the gradual progress of computer hardware and software technologies has a vital role in reducing costs and facilitating the discovery of new small molecules.The use of computer-aided drug design methods allows for the effective usage of prospective therapeutic targets found by genomic and proteomic projects, therefore making the drug discovery process more efficient.In the field of drug design, numerous advanced in silico methods, including docking, molecular dynamics simulation, and a combination of varied sophisticated methodologies, have been used in several studies [9][10][11].

Chemistry
To produce the desired compounds, the synthesis process is initiated by subjecting (E)-Nmethyl-1-(methylthio)-2-nitroethenamine and ninhydrin to a reaction in ethanol at ambient temperature.Subsequently, aliphatic amines were added to the product.The reaction has been carried out for one hour, resulting in the formation of the desired 2-arylamino-dihydroindeno [1,2-b]pyrrol-4(1H)-one (5a-i) products with satisfactory yields ranging from 68% to 87%.The identification of compounds (5a-i) was accomplished by analyzing their Mass, IR, 1 H NMR, and 13 C NMR spectroscopy data, leading to the determination of their structures.The crude product's 1 H and 13 C NMR spectra revealed the presence of two regioisomers.The NMR data for each isomer may be obtained from the mixture of the two isomers, as shown in Fig 2 .As an example, the 1 H and 13 C NMR of 5a revealed a key signal for the NH group (δ 9.85 ppm), multiplets for the aromatic groups (δ 7.73-7.59ppm), a doublet for the aromatic groups (δ 7.30 ppm), two singlets for the OH group (δ 7.22 and 6.37 ppm), a doublet for the aromatic groups (δ 7.15 ppm), an ABX system for CH 2 -benzyl groups (J AB = 16.0Hz, J AX = 8.0 Hz, J BX = 8.0 Hz, δ A = 4.83 ppm, δ B = 4.76 ppm) that are diastereotopic, one singlet for the methyl group (δ 3.35 ppm) for the major regioisomers (87%); and also one multiplet for the NH group (δ 9.46 ppm), multiplets for aromatic and OH groups (δ 7.86-7.70ppm), two doublets for the aromatic groups (δ 7.43 and 7.34 ppm), one singlet for the OH group (δ 6.51 ppm), two AX systems for the CH 2 -benzyl groups as diastereotopic protons (δ 5.39 and 5.19,Δδ = 0.20, 2 J AX = 16.0Hz), and one singlet for the methyl group (δ 2.55 ppm) for the minor regioisomers (13%).The 1 H-decoupled 13 C NMR spectra of compound 5a displayed 16 distinct signals that corresponded with the suggested structure.The mass spectrum of 5a exhibited a molecularion peak at m/z 354.1, which corresponds with the suggested structure.The IR spectra displayed absorption bands at 3472, 3352, and 3218 cm -1 , corresponding to the stretching frequencies of the OH and NH groups.Furthermore, the absorption bands observed at 1733, 1620, 1542, and 1388 cm-1 can correspond to the C = O, NC = C, and NO 2 functional groups, respectively.substitution reaction of aliphatic amine 4 on C-2 of dihydroindeno[2,1-b]pyrrol-8(1H)-one 3', resulting in the elimination of methanethiol.The presence of an electron-withdrawing nitro group at position 3 of the pyrrole ring allows for the acceptance of this procedure while also contributing to the resultant structure's stability via intramolecular hydrogen bonding.Notably, the central C = O, surrounded by two neighboring C = O groups, is likely to possess a higher electrophilic nature.As a result, the formation of compound A as the primary product is expected to be favored over the production of compound A'.

Quantum chemistry through density functional theory calculation
All the selected compounds (5a-i) have been optimized first under gas phase conditions using the B3LYP/6-31++G(d,p) basis set, and their values are displayed in Table 1.
Upon minimizing the energy gradient and optimizing the molecular geometries of the selected compounds, no imaginary frequencies were detected.This suggests that all structures correspond to genuine local minima in the potential energy domain.The optimized structures of these compounds are shown in the Supplementary File.The analysis of molecular orbitals (MO) is of outstanding importance in quantum chemistry and enables a comprehensive understanding of chemical behavior.The most important molecular orbitals within a compound include the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO).These orbitals are used to elucidate various chemical properties, including reactivity, stability, and kinetics.The Supplementary File contains the FMO orbitals for the synthesized compounds.The parameter of hardness (η) serves as a measure to determine the relative hardness or softness of a molecule.The reactivity of a molecule is enhanced as its softness increases.Electronegativity (X) is a fundamental property that quantifies an element's ability to attract electrons.All these properties were estimated at.The compound 5d exhibits the smallest HOMO-LUMO energy gap value 3.9638 eV, indicating high chemical reactivity.It also demonstrates a low hardness value 1.9819 and is the softest molecule among all compounds.Additionally, compound 5d possesses the highest electronegativity value 5.4594, indicating strong electron-attracting capability and making it a better electrophile compared to other compounds.Following 5d, compounds 5e and 5b also display notable reactivity, with energy gap values of 4.0577 eV and 4.0643 eV, respectively.The highest polarizability is related to 5e.After 5e, compounds 5g, 5b, and 5a showed remarkable polarizability with values of 284.54, 284.68, and 285.36, respectively.Table 2 illustrates the energetic parameters of the compounds (5a-i).

Molecular docking studies
Molecular docking was used to assess the binding mechanisms of synthesized compounds with SARS-CoV-2 M pro .The targeting of this protease is important to develop therapeutic approaches against SARS-CoV-2.These targets were selected based on their critical functions in viral protein production; hence, targeting these proteins may provide benefits in virus killing.The crystal structure of the M pro protein identified by the PDB ID: 6W63, has been acquired from the RCSB database.The docking procedure was validated by re-docking the cocrystal ligand into the active pocket of the SARS-CoV-2 M pro protein.The observed rootmean-square deviation (RMSD) value of 1.18 Å indicates a high level of reliability for the docking procedure [14] (Fig 5).
The key amino acid residues in the catalytic site were investigated in docking studies with potent indeno[1,2-b]pyrrol-4(1H)-one derivatives.The results indicated that a considerable portion of the compounds demonstrated substantial binding scores, and among them, compound 5f displayed the most favourable binding energy at -7.28 Kcal/mol.The best  The amino acid residues Glu166, His164, Gly143, Met165, Arg188, Pro52, Tyr54, Asp187, Thr25, Val42, Thr26, Ser144, Asn142, Gln189, His41, Cys145, Leu27, Met49, and Cys44 were involved in both bonding and nonbonding interactions with compound 5f.In summary, compound 5f exhibits four hydrogen bond interactions, encompassing two conventional hydrogen bonds and two carbon hydrogen bonds.Furthermore, it has been observed that compound 5f is accountable for establishing five hydrophobic contacts with the amino acids His41, Cys145, Leu27, Met49, and Cys44 at the active site.Moreover, the amino acid residues Arg188, Pro52, Tyr54, Asp187, Thr25, Val42, Thr26, Ser144, Asn142, and Gln189 were identified as participants in the van der Waals interactions with compound 5f.The docking outcomes of the potent compounds, along with their respective interactions, are delineated in Table 3.

Molecular dynamics simulation
MD simulation of 100 ns was performed for the best protein-ligand complex.

Drug-likeness prediction
Drug likeness refers to the extent of similarity between certain compounds and established drugs.This assessment involves a nuanced balance of molecular and structural properties.Drug similarity assessment is based on various molecular properties such as hydrophobicity, electronic distribution, hydrogen bonding, molecular weight, pharmacophores moiety, bioavailability, reactivity, toxicity, and metabolic stability [15].The Lipinski rule is a widely used method for assessing the solubility and permeability of compounds and thus predicting their suitability as potential drug candidates.According to this rule, compounds that violate Lipinski's rule of five are more likely to have insufficient absorption or permeation.Using the online web server SwissADME [16], our derivatives were analyzed.All of the synthesized compounds (5a-i) do not break the Lipinski rule because their values are within the normal range and their absorption.Also, these compounds (5a-i) are in the best part of the physiochemical space, so they could be thought of as lead compounds.The pharmacokinetic factors showed that the tested compounds (5a-i) are well absorbed by the gastrointestinal system (GI) after being taken by mouth and that P-glycoprotein (P-gp) could move them out of the body.In pharmaceutical chemistry, the pan-assay interference substances (PAINS) structural alerts have been utilized to figure out which parts of the structure are unstable, reactive, and poisonous [17,18].All of the compounds (5a-i) have no alerts in the PAINS descriptions, which is another sign that they could be good drug prospects.The Synthetic Accessibility Score (SA score) serves as a benchmark for assessing the feasibility of synthesizing drug-like molecules.All compounds were found to have a favorable SA score, indicating that they can be easily synthesized, Table 4.

ADMET properties
During the development of novel therapeutic drugs, pharmacological and toxicological knowledge is of the utmost importance.This knowledge not only decreases the duration of drug Similarly, the volume distribution (VD) findings suggest that compounds 5a, 5d, and 5g are not the most limited to blood components and are therefore uniformly distributed across blood and tissue components.Toxicology research is important in drug design since it helps to identify the adverse effects of novel entities on live organisms.The calculation of toxicity indices for all synthesised dihydroindeno[1,2-b]pyrrol-4(1H)-one derivatives revealed that all of the compounds are noncarcinogenic.Moreover, the AMES toxicity assessment revealed that the compounds exhibit no potential for toxicity.Consequently, our synthesized compounds are devoid of any toxic effects.In general, all compounds demonstrated an enhanced ADMET profile, with corresponding values provided in Table 5.

Conclusion
We have effectively synthesized nine novel indeno[1,2-b]pyrrol-4(1H)-one derivatives and identified them using IR, 1 H, and 13 C NMR in this study.The structural geometry was accurately predicted using DFT calculations.Our in silico results show that these compounds have an inhibitory effect against SARS-CoV-2 M pro .In fact, the results showed that compound 5f is more potently inhibitory than the other compounds.Moreover, our result showed that the pharmacokinetics of indeno [1,2-b]pyrrol-4(1H)-one are valuable and provide an informative and promising investigation as effective SARS-CoV-2 M pro inhibitors.Consequently, it hinders the process of viral replication and may serve as a potential therapeutic intervention for combating SARS-CoV-2.Further validation of the chemoinformatics study's findings would require additional in vivo and in vitro investigations.

Computational studies
Density functional theory (DFT).DFT is an effective method for determining electron density and energy parameters in compounds.Its application extends to the determination of atomic, molecular, crystalline, and surface structures and elucidates their interplay.These calculations are carried out with the Gaussian 09W software [22].Using the B3LYP method coupled with a 6-31++G(d,p) basis set, the wavenumbers associated with the vibrations were carefully calculated.The B3LYP functional is widely recognized for its ability to accurately describe harmonic vibrational properties in molecules of modest to moderate size.The positive values of IR frequencies derived from this method imply that the optimized structure has a minimum on the potential energy surface.The generated check files were analyzed using GuassView 6.0.
Molecular docking studies.Molecular docking analysis investigated the interaction between the synthesized compounds and the SARS-CoV-2 M pro protein using Schrodinger's Maestro Molecular Modeling platform.The 3D structure of the protein (PDB: 6W63) [23] was used.All protein preparation for docking was performed, including the use of Protein Preparation Wizard [24] to modify missing residues and ensure structural integrity.GuassView 6.0 helped to visualize the structures of the compounds, which were subsequently converted to.pdb files for the ligprep module.For ligand preparation, the OPLS_2005 force field was used at a pH of 7.0±2 [25].The individual binding sites were defined by a 26 Å grid array created with Glide.Standard accuracy and flexible ligand sampling were used, with the analysis showing 10 poses per ligand.

Molecular dynamic simulation.
A molecular dynamics (MD) simulation was performed with Desmond using the Schroedinger-Maestro interface [26].The results are from the MD simulation performed for the complex following the previous docking phase.An orthorhombic cell filled with water molecules was used, which is representative of the SPC model.Sufficient Na ions were introduced into the system to neutralize the total charge of the complex.The simulation lasted 100 ns and used the NPT ensemble, keeping the number of atoms, the pressure of 1.01325 bar, and the temperature of 300 K constant.The Nose-Hoover chain method with an interval of 1.0 ps was used as the standard thermostat, and the Martyna-Tobias-Klein method with an interval of 2.0 ps was used as the barostat.The interaction diagram of the Maestro simulation was used to analyze the molecular dynamic simulation.

Fig 1 .
Fig 1. Structure of approved antiviral drugs available for treating patients with COVID-19.https://doi.org/10.1371/journal.pone.0299301.g001 Fig 3 displays the complete structures of the synthesized compounds.The mechanism depicted in Fig 4 provides a plausible explanation for synthesizing 2-arylamino-dihydroindeno[1,2-b]pyrrol-4(1H)-one 5 and 2-arylamino-dihydroindeno[2,1-b]pyrrol-8(1H)-one 6.By focusing on the core of ninhydrin, the reaction between the carbonyl group of ninhydrin in positions 1 and 2 and (E)-N-methyl-1-(methylthio)-2-nitroethenamine results in two products.Indeed, this reaction lacks regioselectivity since the formation of the other regioisomer occurred throughout the process.Following path A as shown in Fig 4, the initial step includes the nucleophilic carbon of enamine 2 reacting with the carbonyl group at position 2 of ninhydrin, resulting in the generation of imine A [12,13].The resultant intermediate A undergoes an intramolecular cyclization and an imine-enamine tautomerization to produce diol 3. Subsequently, a nucleophilic substitution occurs at the C-2 position of dihydroindeno[1,2-b]pyrrol-4(3aH)-ones 3 with the aliphatic amine 4.This reaction leads to the formation of intermediate B. Following that, the elimination of methanethiol via intermediate B results in the production of the final product 5.The aliphatic amine 4 undergoes a nucleophilic substitution reaction on C-2 of dihydroindeno[1,2-b]pyrrol-4(3aH)-ones 3, resulting in the formation of intermediate B. This intermediate then eliminates methanethiol to generate the final product 5.Another route is path B in Fig 4, where imine A' is produced when enamine engages in a nucleophilic attack on the carbonyl group in position 1 of ninhydrin.The intermediate A' undergoes an imine-enamine tautomerization and an intramolecular cyclization to produce diol 3'.The desired product 6 is obtained via the nucleophilic

Fig 5 .
Fig 5. Superimposition of the docked ligand (red) and the original ligand (green).https://doi.org/10.1371/journal.pone.0299301.g005 Fig 7 illustrates the importance of the RMSD of both the protein (left Y-axis) and the ligand (right Y-axis) as crucial indicators for verifying the accuracy of the docking geometry.The RMSD of the protein provides detailed insight into its structural conformation during the simulation, while the RMSD of the ligand serves as an indicator of the stability of the ligand in the binding pocket of the protein.The RMSD value fluctuates irregularly at the beginning and reaches stability at 53

Fig 7 .
Fig 7. RMSD values of the protein and the ligand during a MD simulation.https://doi.org/10.1371/journal.pone.0299301.g007 Fig 9 shows the histograms of the interaction fractions of the ligand with each of the key residues of the protein during the simulation time of 100 ns.The hydrogen bonding interactions were shown in green columns, the hydrophobic interactions in purple columns, and the water-bridged hydrogen bonding interactions in blue columns.The most frequent interactions with Phe30 and Asn214 were observed during the entire simulation time.Fig 10 illustrates the properties of compound 5f as determined through a 100-ns MD simulation.Six ligand properties were evaluated: ligand RMSD, radius of gyration (rGyr),

Table 4 . Physicochemical, pharmacokinetics, and medicinal chemistry properties of the compounds (5a-j). MW (g/ mol) HBA HBD TPSA Consensus Log Po/w * MR GI Absorption BBB Permeant P-gp Substrate Lipinski Pfizer PAINS (alert) Bioavailability Score
[21]nces the success rate.ADMET indices, which encompass Absorption, Distribution, Metabolism, Excretion, and Toxicity, are commonly employed to evaluate the nature of a substance.In the case of dihydroindeno[1,2-b]pyrrol-4(1H)-one derivatives, these parameters are determined utilizing the ADMET Lab 2.0 [19].CaCo-2 cells derived from human colonic epithelial cells serve as a common model for the assessment of drug uptake in the human gut.Also, Madin Darby Canine Kidney (MDCK) cells are valuable for the assessment of the rapid permeability of drug molecules due to their shortened growth cycle compared to CaCo-2 cells[20].The CaCo-2 cell permeability results for our synthesized compounds fell within an acceptable range, suggesting that these compounds possess exceptional intestinal absorption.Moreover, all compounds displayed positive MDCK cell permeability, indicating a higher probability of elimination through kidney cells.The results indicated that all compounds exhibited characteristics suggestive of being both PGP-substrates and PGP-inhibitors in relation to plasma glycoprotein (PGP) interactions.Based on the human intestinal absorption (HIA) values, it can be inferred that all compounds have a favorable likelihood of being absorbed through the intestinal membrane.Compounds exhibiting CBrain/CBlood values exceeding 1 are classified as having central nervous system (CNS) activity, whereas compounds with CBrain/CBlood values below 1 are classified as CNS inactive.Compounds possessing central nervous system (CNS) activity have the ability to cross the Blood-Brain Barrier (BBB) and result in side effects on the central nervous system[21].According to the data presented in Table5, all of our compounds have CBrain/CBlood values below 1, indicating that they are not capable of crossing the BBB.Consequently, our synthe- MW: Molecular Weight; HBA: Num.H-Bond Acceptors; HBD: Num.H-Bond Donors; MR: Molar Refractivity; TPSA: Topological Polar Surface Area; P-M: Poor-Moderate; P: Poor; GI: Gastrointestinal; BBB: Blood-Brain Barrier; P-gp: P Glycoprotein.https://doi.org/10.1371/journal.pone.0299301.t004developmentbut also sized compounds are devoid of neurotoxicity.The percentage representation of the plasma protein binding model determines the extent to which a compound is strongly bound to blood carrier proteins.The plasma protein binding values for compounds 5a, 5d, and 5g exceed 94%, suggesting that our synthesized derivatives of dihydroindeno[1,2-b]pyrrol-4(1H)-one possess sufficient bioavailability and are unlikely to exhibit strong binding to blood carrier proteins.